We report on a paper-based 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay for a simple, inexpensive, low reagent and sample consumption and high throughput analysis of antioxidant activity. The paper-based device was fabricated using a lamination method to create a 5-mm in diameter circular test zone that was embedded with a DPPH reagent. The analysis was carried out in one-step by dropping an antioxidant/sample onto the test zone. After reduction by the antioxidant, the DPPH radicals become stable DPPH molecules, resulting in a change in color from deep violet to pale yellow. The violet color intensity of DPPH was inversely proportional to the antioxidant activity of the samples, and was measured using imaging software. A high precision and a low limit of detection were found in the analysis of six standard antioxidants including gallic acid, trolox, ascorbic acid, caffeic acid, vanilliic acid and quercetin. The device was then validated against the traditional spectrophotometric DPPH assay by analyzing the antioxidant activity of 7 tea samples. The results showed no significant difference for gallic acid equivalent for all 7 samples obtained from the two methods at the 95% confidence level, indicating that the developed method was reliable for antioxidant activity analysis of real samples. Finally, the paper-based DPPH device was found to be stable over 10 days when stored in a refrigerator (2 - 4°C), making it an easy-to-use device for end-users.

We report a simple, low-cost, one-step fabrication method for microfluidic paper-based analytical devices (μPAD) using only polystyrene and a patterned screen. The polystyrene solution applied through the screen penetrates through the paper, forming a three-dimensional hydrophobic barrier, defining a hydrophilic analysis zone. The optimal polystyrene concentration and paper types were first investigated. Adjusting polystyrene concentration allows for various types of paper to be used for successful device fabrication. Using an optimized polystyrene concentration with Whatman#4 filter paper, a linear relationship was found to exist between the design width and the printed width. The smallest hydrophilic channel and hydrophobic barrier that can be obtained are 670 ± 50 μm and 380 ± 40 μm, respectively. High device-to-device fabrication reproducibility was achieved yielding a relative standard deviation (%RSD) in the range of 1.12-2.54% (n = 64) of the measured diameter of the well-shaped fabricated test zones with a designed diameter of 5 and 7 mm. To demonstrate the significance of the fabricated μPAD, distance-based and well-based paper devices were constructed for the analysis of H2O2 and antioxidant activity, respectively. The analysis of H2O2 in real samples using distance-based measurement with CeO2 nanoparticles as the colorimetric agent produced the same results at 95% confidence level, as those obtained using KMnO4 titration. A proof-of-concept antioxidant activity determination based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was also demonstrated. The results verify that the polymer screen-printing method can be used as an alternative method for μPAD fabrication.

We report the first use of a paper-based device coated with nanoceria as a simple, low-cost and rapid detection platform for the analysis of organophosphate (OP) pesticides using an enzyme inhibition assay with acetylcholinesterase (AChE) and choline oxidase (ChOX). In the presence of acetylcholine, AChE and ChOX catalyze the formation of H2O2, which is detected colorimetrically by a nanoceria-coated device resulting in the formation of a yellow color. After incubation with OP pesticides, the AChE activity was inhibited, producing less H2O2, and a reduction in the yellow intensity. The assay is able to analyze OP pesticides without the use of sophisticated instruments and gives detection limits of 18 ng mL(-1) and 5.3 ng mL(-1) for methyl-paraoxon and chlorpyrifos-oxon, respectively. The developed method was successfully applied to detect methyl-paraoxon in spiked vegetables (cabbage) and a dried seafood product (dried green mussel), obtaining ∼95% recovery values for both sample types. The spiked samples were also analyzed using LC-MS/MS as a comparison to the developed method and similar values were obtained, indicating that the developed method gives accurate results and is suitable for OP analysis in real samples.